1. Field of the Invention
The invention lies in the art of biomedical, scientific instrumentation. More specifically the invention relates to cytometers and instruments for high speed identification and sorting of cells, organelles and chromosomes. In particular the invention discloses an improved cytometer event deflection and sorting apparatus and method.
2. Description of the Prior Art
Various techniques of flow cytometry have been employed over the last quarter century from an initial effort to count particulate matter in a fluid environment, subsequently to size particles and more recently to quickly quantify multiple chemical, physical or structural properties of cells and cellular composites of inhomogeneous populations. The first such effort related to counting individual red cells in a liquid suspension forced through a capillary glass tube on a microscope stage. Problems encountered by such means involved standardizing capillary tubes, assuring proper focus, maintaining even flow and obtaining appropriately sensitive photoelectric apparatus to accomplish an accurate count.
Some of these problems were resolved by injecting the particle suspension into a laminar sheath flow of fluid, which flow surrounded and aligned the particles, and thereby virtually eliminated large particle blockage and coated the particle stream. Particle count by such means was accomplished by detecting the variation of electrical characteristic of the path through the laminar flow caused by the inclusion or exclusion of cellular matter therein. In addition particle sizing could be accomplished because pulse amplitude width was related to particle volume, and could be evaluated by pulse-height analyzers or nuclear pulse amplifiers. Photoelectric counting was later introduced. Subsequent cytometric application utilized spectrophotometry to quantify cellular constituents or alternatively to clarify cellular constituents via multiple simultaneous measurements of different cellular features, through UV absorption and photon scattering.
All the foregoing systems required a suspension of cells to pass through a constricted channel traversed by a beam of light orthogonal to said channel in which light intensity varied dependent upon position of the cell in the channel. Another possible variation, however, directed the light beam parallel to the flow and made calculations based on light scatter. Florescence at variable wavelengths or absorption characteristics were later used to characterize DNA and RNA constituents in the flow orthogonal to the illuminating beam.
Later cytometric improvements involved pneumatic, hydraulic and electrostatic techniques to separate cells from a flow after photometric or electrical sensing. A following fluid switch cell sorter diverted a stream by means of a sonic transducer that converted laminar flow to turbulent flow.
More recent efforts utilize a sheath fluid flow chamber to which is centrally added a fluid flow of sample body cells or organelles in aqueous suspension. The flow chamber is vibrated at high frequency by a piezoelectric transducer which causes a sheath stream jet exiting the flow chamber with samples to break into discrete droplets from an exit point of the flow chamber. Upon exiting the flow chamber the jet and discrete droplets pass through electrical charging means that charge each droplet either positively or negatively as determined by laser identification of samples or events in the sheath flow prior to droplet formation. The charged droplets then pass through a pair of vertical plates, one charged at a negative voltage and the other at a positive voltage. The positively charged droplets shift stream toward the negative plate and the negative droplets shift stream toward the positive plate. Uncharged droplets continue in a straight line out of the flow chamber to a collector tube below.
Although the foregoing electronic charging of droplets allows for sorting of particles with two attributes by positive or negative charging, there remains long standing need for identification of more physical or chemical characteristics than presently exist in the art, and therefor more accurate sorting of events than is permissible with state of the art deflecting mechanisms. As described above traditional deflection plates are rectangular shaped conductive plates of opposite charge at .+-.3000 volts. The plates are ungrounded, uncovered and unshielded, thereby subjecting an operator to possible unwarranted and hazardous electrical shock. In addition, being unshielded, the strong electric field of the deflection plates may affect charging of the charged flow stream of event droplets at the point of inducing the charge which can affect the charge and distort accurate deflection of the stream. Furthermore, the virtually straight lines of electric force from positive plate to negative plate and perpendicular to the charged stream flow does not allow for accurate stream deflection. Charged droplets can easily and without hindrance flow along parallel equipotential lines between the charged plates.
If the electric field were not only perpendicular to the stream flow, but also doubly curved back on itself by each oppositely charged field plate, the electric field would possess a plurality of force vectors generally flowing in opposed directions, creating a focusing effect of the charged droplet stream. By putting a ground plate on each oppositely charged deflection plate, in accordance with the invention an increased and oppositely curved electric field with multidirectional force vectors is obtained yielding a much more potent focusing force field than is possible with the prior art unidirection electric field.